4.2- Effect of BHRF1 on cell metabolism: Flux Balance Analysis
Metabolism of animal cells growing in high glucose concentration media is characterized by their high rates of glycolysis fluxes. Glucose metabolism of aerobic microorganisms yield pyruvate, which is partially converted into acetyl-CoA and then can be completely oxidized to CO2 and H2O in TCA cycle. However, in mammalian cell lines, pyruvate is primarily converted into lactate (Martínez-Monge et al., 2018a). Generation and accumulation of large amounts of lactate, since glucose cannot be completely oxidized, is the major consequence of such high glycolytic fluxes, leading to an unbalanced metabolism. This metabolic behavior has been observed in many hybridoma cell lines, regardless of the level of dissolved oxygen in culture. Lactate generation from pyruvate seems to be necessary to fulfill the NADH regeneration requirements in the cytoplasm (Mulukutla et al., 2012), due to the limiting transport rates of NADH into mitochondria, where can also be regenerated. Even though pyruvate conversion to lactate is a much less energetically efficient process than its oxidation in the Krebs cycle (Martínez et al. 2013).
Flux Balance Analysis (FBA) is a usefulness tool to obtain more information about the redistribution of the internal metabolic fluxes and could help to generate some hypothesis about the effects on metabolism generated by BHRF1. Figures 4 and 5 show the distribution of the metabolic fluxes using the genome-derived model for Mus musculus detailed in the Calculations section. Metabolic flux balances were conducted using the data obtained from the bioreactor cultures.
FBA shows a deregulated metabolism in both cell lines, characterized by high glycolytic fluxes and the consequent lactate generation and secretion. However, glycolytic fluxes were reduced by 54% (bottom part of glycolysis as a reference) due to the reduction in glucose consumption (51%) in KB26.5-BHRF1. Lactate generation flux was dropped to more than 60%. Despite the lower fluxes in glycolysis presented by KB26.5-BHRF1 strain respect to KB26.5, the rate of carbon influx from cytoplasm to mitochondria through pyruvate was 14% higher (320 versus 274 nmol/(mg·h)). In other words, when calculating the fluxes in mass units (mg metabolite/(mg DCW ·h)) only 13% of the total glucose consumed is entered to the mitochondria in KB26.5 cells, whereas it increases up to 32% in the engineered KB26.5-BHRF1. When performing similar calculations to estimate the lactate generation ratio regardless the glucose consumption, lactate formation represents about 87% and 69% of the consumed glucose in KB26.5 and KB26.5-BHRF1, respectively.
Lactate generation in mammalian cell cultures is a well-known issue that has been extensively studied (Hartley et al., 2018; Zagari et al., 2013). At present, the most accepted hypothesis of why lactate is generated argues in the regeneration of the reducing power (NADH) in the cytoplasm, due to the high glycolytic fluxes (Hartley et al., 2018; Zheng, 2012). To this end, there are two ways to regenerate NADH into the cytoplasm: 1) pyruvate conversion to lactate and 2) Malate-Aspartate Shuttle (Mulukutla et al., 2012). The interesting point is that Malate-Aspartate Shuttle allows not only to regenerate NADH but also to increase TCA cycle fluxes (importing malate), allowing then to generate energy in form of ATP. On the other side, lactate generation provokes the total loss of both carbon source and ATP generation. Then, the reason for lactate generation could be found in a flux limit of Malate-Aspartate Shuttle, leading the cells to generate lactate and display this wasteful metabolism.
In the glucose breakdown to two pyruvate molecules, two molecules of ATP and two of NADH are generated. Since the inner mitochondrial membrane is impermeable to NADH, Malate-Aspartate Shuttle works as an indirect transport system. It has been reported that the flux through this transport occurs at lower rates than glycolysis (Schantz et al., 1986), and the increased LDH activity is due to the inability of transporting NADH through the shuttle at the same rates in which is generated. Under conditions of increased cellular energy demand, higher glycolytic fluxes are observed and, consequently, NADH production rates increases proportionally. Such increase in NAD+ regeneration needs is compensated by higher LDH activity in the cytosol, but not differences in the Malate-Aspartate Shuttle fluxes are observed (Robergs et al., 2004).
In this case, FBA shows that the rate of transport of reduction power from cytoplasm to mitochondria through Malate-Aspartate Shuttle seems to be a bit higher in KB26.5-BHRF1 than in KB26.5, showing a 19% of increase, with a rate of 228 versus 192 nmol/(mg·h) in the aspartate-glutamate mitochondrial symport transport. However, in both cases the high glycolytic fluxes lead the cells to generate lactate, as the Malate-Aspartate shuttle seems to not be enough powerful to couple with the cytoplasmic NADH regeneration. In addition, a slight increase in pathways related with the biomass formation is observed in KB26.5-BHRF1, due to the higher growth rate; as Pentose Phosphate Pathway to generate nucleotides or citrate export from the mitochondria to lipids synthesis.
Due to the high growth rate in KB26.5-BHRF1, an increase in the TCA fluxes could be expected, to generate more energy and therefore biomass. However, comparing TCA cycles for both cell lines no significant increase is observed, what is in accordance with the similar oxygen consumption rate (Table 5 ). What it is clear from the FBA performed is the evident collapse between glycolysis and TCA pathways in both cases, as the high glucose uptake rate collapses the transport of NADH to mitochondria, thus NADH should be regenerated into the cytoplasm using pyruvate by means of lactate dehydrogenase.
The reduction of the glycolytic pathway in KB26.5-BHRF1 involves a reduction of ATP production in the cytoplasm, as well as a reduction of NADH formation. Consequently, the needs for oxidizing the NADH formed in the cytoplasm also decreased and, together with the slightly higher rates related to Malate-Aspartate Shuttle, yielded to a reduction of the lactate formation. To further discuss the results, an analysis of the synthesis and consumption of ATP was performed. Figure 6 shows the distribution of the ATP formation (in regard to the reaction in which ATP is generated) and its consumption.
The results show a significant reduction in the total ATP generation and, as a consequence, ATP consumption in KB26.5-BHRF1 compared with the parental cell line. As pointed out above, higher amounts of ATP were synthetized along glycolysis in parental KB26.5 (proportionally to glycolytic fluxes increase), but also an increase in the oxidative phosphorylation pathway is observed, being very important for the discussion for its high capacity of ATP generation. Regarding the ATP consumption, KB26.5-BHRF1 needs more ATP to generate more biomass, which also includes other pathways as lipids and nucleotide synthesis. The most interesting part, and the key fact of this analysis, is the huge difference in ATP requirements for cell maintenance. One possible explanation of this maintenance reduction could be the higher efficient substrate consumption and specially the lactate generation reduction of KB26.5-BHRF1. As recently pointed out by Buchsteiner et al. (2018), the energy requirements in lower lactate production cell lines may decrease due to the lower energy requirements for maintaining ion gradients.
The results presented here suggest that the engineered KB26.5-BHRF1 hybridoma cell line somehow have altered such metabolism. BHRF1 is an antiapoptotic protein located in the inner mitochondrial membrane (Milian et al. 2015). Due to this fact, BHRF1 could be somehow affecting carbon and NADH/NAD+ transport between cytoplasm and mitochondria. In fact, most of the antiapoptotic genes reported in literature are known to be bind at the mitochondrial membrane, regulating the apoptosis through modulation of the mitochondrial permeability, but also playing and important role in the metabolic processes of mitochondria (Majors et al., 2007). Dorai et al. (2009) reported the effect of two antiapoptotic genes in the metabolism of CHO, showing an important reduction in the final lactate concentration due to the lactate consumption during the culture. In addition, engineered cells showed a more efficient nutrient consumption profile and less by-products generation, as ammonia or alanine, as observed in this study.